U.S. patent number 5,989,526 [Application Number 09/019,261] was granted by the patent office on 1999-11-23 for tooth bleaching.
This patent grant is currently assigned to Colgate-Palmolive Company, Novo Nordisk A/S. Invention is credited to Dorrit Aaslyng, Abdul Gaffar, Sahar F. Smith, Rie Tsuchiya.
United States Patent |
5,989,526 |
Aaslyng , et al. |
November 23, 1999 |
Tooth bleaching
Abstract
The present invention provides safe products for bleaching
teeth. Oral compositions comprising at least one oxidoreductase
fulfil said criteria without the presence of peroxide or with only
very little peroxide present. The invention also relates to an oral
care product comprising the oral composition of the invention, a
method of bleaching teeth, a method for using said oral care
products and the use of oxidoreductases for oxidation of teeth
stains.
Inventors: |
Aaslyng; Dorrit (V.ae
butted.rl.o slashed.se, DK), Tsuchiya; Rie (Birkerod,
DK), Gaffar; Abdul (Princeton, NJ), Smith; Sahar
F. (Bordenton, NJ) |
Assignee: |
Novo Nordisk A/S (Bagsv.ae
butted.rd, DK)
Colgate-Palmolive Company (New York, NY)
|
Family
ID: |
26064917 |
Appl.
No.: |
09/019,261 |
Filed: |
February 5, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCTDK9600350 |
Aug 19, 1996 |
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Foreign Application Priority Data
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Aug 18, 1995 [DK] |
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0926/95 |
Sep 20, 1995 [DK] |
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1048/95 |
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Current U.S.
Class: |
424/50;
424/49 |
Current CPC
Class: |
A61Q
11/00 (20130101); A61K 8/66 (20130101) |
Current International
Class: |
A61K
8/66 (20060101); A61K 8/30 (20060101); A61Q
11/00 (20060101); A61K 007/16 (); A61K 007/28 ();
A61K 007/30 () |
Field of
Search: |
;424/50,51 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: Rose; Shep K.
Attorney, Agent or Firm: Zelson, Esq.; Steve T. Green, Esq.;
Reza
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. 119 of Danish
application serial numbers 0926/95 filed on Aug. 18, 1995 and
1048/95 filed on Sep. 20, 1995 and priority under 35 U.S.C. 120
which is a continuation of application serial no. PCT/DK96/00350
filed on Aug. 19, 1996 in the PCT, the contents of which are fully
incorporated herein by reference.
Claims
We claim:
1. An oral care product comprising an enzyme selected from the
group consisting of laccase, catechol oxidase, bilirubin oxidase,
mono-phenol mono-oxygenase, and combinations of any of the
foregoing.
2. The oral care product of claim 1, wherein the laccase is derived
from Polyporus sp.
3. The oral care product of claim 2, wherein the laccase is derived
from a strain of Polyporus pinsitus.
4. The oral care product of claim 1, wherein the laccase is derived
from Myceliophthora.
5. The oral care product of claim 4, wherein the laccase is derived
from a strain of M. thermophilia.
6. The oral care product of claim 1, wherein the laccase is derived
from Scytalidium.
7. The oral care product of claim 6, wherein the laccase is derived
from a strain of S. thermophilium.
8. The oral care product of claim 1, further comprising a redox
mediator.
9. The oral care product of claim 8, wherein the mediator is
selected from the group consisting of
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) and
10-propionic acid-phenothiazine (PPT).
10. The oral care product of claim 1, wherein the enzyme acts on
non-cariogenic substrates.
11. The oral care product of claim 1, wherein the enzyme is
substantially active between about pH 5.0 and about pH 9.0.
12. The oral care product of claim 1, further comprising an enzyme
selected from the group consisting of a protease, an amylase, a
dextranase, a mutanase, a lipase, and combinations of any of the
foregoing.
13. The oral care product of claim 1, wherein said product
comprises an anti-microbial activity.
14. The oral care product of claim 1, wherein said product is
selected from the group consisting of a dentifrice, a toothpaste, a
tooth powder, a mouth wash, a pre-rinse, and a denture cleaning
agent.
15. A method for bleaching teeth, comprising contacting said teeth
with the oral care product of claim 1 under conditions suitable for
bleaching teeth .
Description
FIELD OF THE INVENTION
The present invention relates to an oral composition for bleaching
teeth without the presence of peroxide or comprising only very
little peroxide, an oral care product comprising the oral
composition of the invention, a method of bleaching teeth, a method
for using said oral care product and the use of oxidoreductases for
oxidation of teeth stains.
BACKGROUND OF THE INVENTION
Most people want to have a mouth full of dazzling white teeth, as
the teeth constitute an important part of the overall picture of
the human face especially when smiling. In contrast hereto strongly
discoloured teeth entail an unhealthy and in certain cases even
repellent "look".
For many years, crowns or dentures were seen as the only means for
avoiding e.g. the yellowing of teeth coming with age,
discolouration of teeth due to long term intake of the antibiotic
tetracycline during childhood, or the yellowish brown to black
discolouration of teeth as a consequence of coffee drinking,
tobacco smoking/chewing etc.
Even though techniques for bleaching teeth have been known for many
years, tooth bleaching has never been used extensively, until
recently.
The first published tooth bleaching technique used oxalic acid as
the bleaching agent and was reported by Chaple in the 1877. Soon
after, the first report of peroxide used as a bleaching agent was
published by Harlan in 1884. Even though quite a number of
bleaching agents have been suggested since then peroxide is today
still the most commonly used agent.
The structure of teeth
A mammal tooth is chiefly composed of four main constituents namely
the "cementum", the "dental pulp", the "dentin", and the
"enamel".
The "cementum" is bonelike tissue forming the outer surface of the
root of the tooth. The "dental pulp" consists of sensitive tissue
containing arteries, veins, and lymphatic and nerve tissue. The
"dentin" which surrounds the dental pulp forms the major part of
the tooth. The dentin is dense bonelike calcareous tissue. On the
outside of the tooth there is a hard porous layer composed of
hydroxyapatite mineral crystals having a natural opaque white or
off-white colour. This outer porous layer is called the
"enamel".
Tooth discolouration
Tooth discolouration can be caused by a variety of intrinsic and/or
extrinsic influences. In general stains are divided into two main
categories: 1) extrinsic stains and 2) intrinsic strains.
1) Extrinsic stains
Extrinsic stains are mainly caused by the daily intake of
substances, such as foods and beverages through the mouth, and/or
the use of tobacco products etc. These substances tend to adhere to
the enamel's hydoxyapatite structure and hereby discolour the teeth
and/or reduce the whiteness of the teeth.
Over a period of years extrinsic stains may penetrate the enamel
layer and gradually give intrinsic discolourations.
2) Intrinsic stains
Intrinsic stains are the term used for stains which have penetrated
the tooth structure (i.e. discolouration within the tooth matrix).
Such stains can arise as described above or be caused by agents
including haematological agents and certain drugs, or be due to
dental pulp necrosis or developmental abnormalities.
For instance, degradation products from the body may cause
discolorations. Excessive intake of tetracycline or fluoride during
a long period of illness has been found to release degradation
products into the dentinal tubules during the development of tooth
enamel causing some degree of discolouration. The severity of such
discolouration depends on the time and duration of intake of the
medicine.
Further, dental pulp necrosis entails Haemorrhagic discolorations
and is a result of blood degradation. If the pulp necrosis is
caused by e.g. caries degradation of proteins the tooth/teeth will
become greyish-brown.
In the case of traumatic pulp death the tooth will turn
yellow-brown. It is believed that such a discolouration is caused
by haemolysis of red blood cells entering the dentinal tubules.
Tooth bleaching
Before conducting tooth bleaching it is important to assess the
type of stain, as different types of stains need different
approaches and/or bleaching agents.
Certain extrinsic stains, which occur on the surface or subsurface
of the teeth, can be removed by regular intense mechanical brushing
of the teeth with cleansing agents containing abrasives and
surfactants. However, not all extrinsic stains can be removed this
way and require bleaching agents which inhibit non-enzymatic
browning reactions.
Intrinsic stains are located in the tooth matrix and cannot be
removed or prevented by intense mechanical brushing of the teeth.
Removal of such discolourations requires bleaching agents capable
of penetrating into the teeth structure. Hydrogen peroxide is an
example of such an agent, which can be used for both extrinsic and
intrinsic stains.
Hydrogen peroxide can be used for many types of stains e.g. stains
residing in the dentin, such as stains caused by tetracycline.
Even though hydrochloric acid is not regarded as being a bleaching
agent it is known to be capable of removing stains caused by
fluorosis, as it dissolves the surface of the teeth.
However, the use of such agents can inflict severe tooth damage or
at least irritation in the oral cavity. Consequently, such agents
are for safety reasons not suitable for "home-use" by the private
consumer and should only be used precautiously by
professionals.
Bleaching techniques
Bleaching techniques are usually divided into two main
categories:
a) non-vital bleaching techniques, and
b) vital bleaching techniques.
a) Non-vital bleaching
The non-vital techniques give the most effective results but also
have the greatest potential hazard. One non-vital bleaching
technique uses sodium perborate and 35% hydrogen peroxide as the
active ingredient.
b) Vital bleaching
Products sold for vital bleaching techniques can be divided into
three main groups a) "in-office" bleaching products, b) dentist
prescribed, home applied bleaching products, and c)
over-the-counter bleaching kits.
For further information concerning the categorisation of tooth
bleaching products and techniques we refer to Van B. Haywood,
(1992), Periodontology and Restorative Dentistry, p. 142-149.
One of the most commonly used "in-office"-techniques combines the
use 30% hydrogen peroxide with heat and light treatment to speed up
the oxidation reaction (i.e. the removal of stains).
Another method, using a "dentist prescribed,
home-applied"-bleaching product, involves the use of 10% urea
peroxide (carbamide peroxide). The teeth are bleached in a mouth
tray, containing the bleaching agent, placed upon the teeth of the
patient.
Over-the-counter kits which can be used for bleaching teeth include
products such as toothpastes and mouth washes having from 3% to 6%
hydrogen peroxide and are sold directly to the consumer, without
prescription by a dentist.
Comments to prior art
Most of the above mentioned prior art methods/techniques involve
the use of peroxides and/or other agents in concentrations which
are not safe for "home-use" by the private consumer due to the risk
of damaging the teeth and/or the oral tissue.
Furthermore, effective concentrations of e.g. hydrogen peroxide
exceed the allowed limits in certain countries.
Products comprising low concentration of bleaching agents, such as
hydrogen peroxide, are considered to have slow bleaching
effect.
Therefore, there is a need for providing safe tooth bleaching
compositions, which do not comprise harmful concentrations of
peroxide and/or other hazardous agents. It is further desirable
that such tooth bleaching compositions can be used as components in
conventional oral care products for "home-use" by the private
consumer.
SUMMARY OF THE INVENTION
The object of the present invention is to provide safe tooth
bleaching products, which do only comprise very low concentrations
of peroxides or/and other hazardous bleaching agents.
The present inventors have surprisingly found that tooth bleaching
compositions comprising at least one oxidoreductase have a good
bleaching effect without causing the problems that high
concentrations of peroxides and other agents may cause.
Consequently, in the first aspect the object of the present
invention is to provide an oral composition for bleaching teeth
comprising at least one oxidoreductase, such as a laccase or a
related enzyme, and/or an oxidase and/or a peroxidase.
In the second aspect the invention relates to an oral care product,
comprising an oral composition for bleaching teeth of the
invention.
The invention also relates to a method for bleaching discoloured or
stained teeth, and a method for using an oral composition or an
oral care product in the oral cavity.
Finally the invention relates to the use of oxidoreductases for
vital and non-vital bleaching of teeth.
DETAILED DESCRIPTION OF THE INVENTION
The object of the present invention is to provide safe tooth
bleaching products which do only comprise very low concentrations
of peroxides or/and other hazardous agents.
In an embodiment of the invention it is even possible to omit
peroxide and/or other equivalent agents completely from the
composition.
In the context of the present invention the term "bleaching" of
teeth is equivalent with the terms "whitening" or "brightening" of
teeth.
The term "low concentrations" of e.g. peroxide means concentration
of from 0% (no peroxide present) to about 1% peroxide, calculated
on the basis of the weight of final oral composition or oral care
product.
All concentrations mentioned in the connection with the present
invention are calculated in weight per cent.
Safe oral compositions and oral care products of the invention may
comprise from 0% to 0.5% peroxide, such as less than about 0.3%,
which may be about 0.1% peroxide.
Preferably the concentration of peroxide lies from 0% to 1%.
It is to be understood that even though less than 1% of peroxide is
needed for obtaining a tooth bleaching effect of the 9invention, it
is contemplated to add concentration of peroxide that is higher
than 1%, such as 3% or 6% and even 10% calculated on the basis of
the weight of the final oral composition or oral care product.
However, if peroxide is present in higher concentrations it may
damage the oral tissue. Further, the stain oxidising enzyme may be
inactivated at such concentrations.
The use of oral care product comprising an oral composition of the
invention facilitates the bleaching of teeth. For instance oral
care products of the invention in the form of a toothpaste or a
mouth wash make it possible to incorporate tooth bleaching as a
natural part of the daily teeth cleaning and/or mouth rinsing at
home.
The present inventors have surprisingly found that safe tooth
bleaching compositions can be provided by adding an enzyme or
enzyme system capable of oxidising the teeth stains.
In the first aspect the invention relates to an oral composition
for bleaching teeth comprising at least one enzyme within the group
of oxidoreductases as the active bleaching ingredient(s).
Oxidoreductases (i.e. enzymes classified under the Enzyme
Classification number E.C. 1 (Oxidoreductases) in accordance with
the Recommendations (1992) of the International Union of
Biochemistry and Molecular Biology (IUBMB)) which are enzymes
catalysing oxidoreductions.
Within the group of oxidoreductases enzymes are preferred which
catalyse the oxidation of a substrate (an electron or hydrogen
donor) by acting on oxygen (O.sub.2) and/or a peroxide as the
acceptor. Such enzymes include enzymes classified within the enzyme
classes comprising oxidases including E.C. 1.1.3. E.C. 1.2.3, E.C.
1.3.3, E.C. 1.4.3, E.C. 1.5.3, E.C. 1.7.3, E.C. 1.8.3, E.C. 1.9.3,
laccases and related enzymes comprised in E.C. 1.10.3 and
peroxidases in E.C. 1.11.
In the case of an enzyme acting on oxygen (O.sub.2) as the
acceptor, said oxygen may be molecular oxygen supplied by the
air.
If a peroxide, for instance H.sub.2 O.sub.2 or H.sub.2 O.sub.2
-generation compounds such as perborat and percarbonate, is added
to the oral composition or oral care product it will be added in
the above mentioned concentrations.
Enzymes' or enzyme systems' ability to fulfil the above criteria of
oxidising teeth stains can be assayed by using the approach
described below in the "Materials and Methods"-section.
According to the invention three types of oxidoreductases are
specifically contemplated:
a) Laccases or related enzymes, which act on molecular oxygen and
yield water (H.sub.2 O) without any need for peroxide (e.g. H.sub.2
O.sub.2),
b) Oxidases, which acts on molecular oxygen (O.sub.2) and yield
peroxide (H.sub.2 O.sub.2), and
c) Peroxidases, which act on peroxide (e.g. H.sub.2 O.sub.2) and
yield water (H.sub.2 O).
Also enzyme systems which comprise a combination of the three types
of enzymes are contemplated according to the invention. The enzyme
systems may e.g. consist of a laccase and an oxidase; a laccase and
a peroxidase; a laccase and an oxidase and a peroxidase; or an
oxidase and a peroxidase.
Preferred are the below mentioned enzymes, especially recombinant
and/or substantially purified enzymes.
In the context of this invention "laccases and related enzymes"
include enzymes comprised by the enzyme classification E.C.
1.10.3.2 (laccases) and catechol oxidase enzymes comprised by E.C.
1.10.3.1, bilirubin oxidase enzymes comprised by the enzyme
classification E.C. 1.3.3.5 and mono-phenol mono-oxygenase enzymes
comprised by the enzyme classification E.C. 1.14.99.1.
Preferably, the laccase employed is derived from a strain of
Polyporus sp., in particular a strain of Polyporus pinsitus or
Polyporus versicolor, or a strain of Myceliophthora sp., e.g. M.
thermophila or a strain of Rhizoctonia sp., in particular a strain
of Rhizoctonia praticola or Rhizoctonia solani, or a strain of a
Rhus sp., in particular Rhus vernicifera.
In specific embodiments of the invention the oxidoreductase is a
laccase, such as a Polyporus sp. laccase especially the Polyporus
pinisitus laccase (also called Trametes villosa laccase) described
in WO 96/00290 (from Novo Nordisk Biotec, inc.) or a Myceliophthora
sp. laccase especially the Myceliophthora thermophila laccase
described in WO 95/33836 (from Novo Nordisk Biotech inc).
Further, the laccase may be a Scytalidium sp. laccase, such as the
S. thermophilium laccase described in WO 95/33837 (from Novo
Nordisk Biotech inc.) or a Pyticularia sp. laccase, such as the
Pyricularia oryzae laccase which can be purchased from SIGMA under
the trade name SIGMA no. L5510, or a Coprinus sp. laccase, such as
a C. cinereus laccase, especially a C. cinereus IFO 30116 laccase,
or a Rhizoctonia sp. laccase, such as a Rh. solani laccase,
especially the neutral Rh. solani laccase described WO 95/07988
(from Novo Nordisk A/S) having a pH optimum in the range from 6.0
to 8.5.
The laccase may also be derived from a fungi such as Collybia,
Fomes, Lentinus, Pleurotus, Aspergillus, Neurospora, Podospora,
Phlebia, e.g. P. radiata (WO 92/01046), Coriolus sp., e.g. C.
hirsitus (JP 2-238885), or Botrytis.
Bilirubin oxidase may preferably be derived from a strain of
Myrothecium sp., such as M. verrucaria.
Within the group of peroxidases classified under the Enzyme
Classification number E.C. 1.11 (peroxidases) peroxidases
(1.11.1.7) are especially contemplated.
The peroxidase of the invention may be derived from plants (e.g.
horseradish peroxidase) or micro-organisms including fungi and
bacteria such as a strain of Coprinus sp., such as Coprinus
cinereus or Coprinus macrorhizus, or bacteria such as Bacillus,
such as Bacillus pumilus.
In another embodiment of the invention the oxidoreductase is a
peroxidase, such as a Coprinus sp. peroxidase, such as the C.
cinereus peroxidase, such as C. cinereus IF08371 or variants
thereof described in WO 95/10602 (from Novo Nordisk A/S) being very
stable in the presence of hydrogen peroxide, or a haloperoxidase
originating from a strain of Curvularia sp. such as a C.
verrruculosa haloperoxidase, in particular C. verruculosa CBS
147.63.
Oxidases which are contemplated include glucose oxidase (E.C.
1.1.3.4), hexose oxidase (E.C. 1.1.3.5), L-amino-acid oxidase (E.C.
1.4.3.2), xylitol oxidase, galactose oxidase (E.C. 1.1.3.9),
pyranose oxidase (E.C. 1.1.3.10), alcohol oxidase (E.C.
1.1.3.13).
If a L-amino acid oxidase is used it may be derived from a
Trichoderma sp. such as Trichoderma harzianum, such as the L-amino
acid oxidase described in WO 94/25574 (from Novo Nordisk A/S), or
Trichoderma viride.
A suitable glucose oxidase may originate from Aspergillus sp., such
as a strain of Aspergillus niger, or from a strain of Cladosporium
sp. in particular Cladosporium oxysporum, especially Cl. oxysporum
CBS 163 described in WO 95/29996 (from Novo Nordisk A/S).
Hexose oxidases from the red sea-weed Chondrus crispus (commonly
known as Irish moss)(Sullivan and Ikawa, (1973), Biochim. Biophys.
Acts, 309, p. 11-22; Ikawa, (1982), Meth. in Enzymol. 89,
carbohydrate metabolism part D, 145-149) oxidises a broad spectrum
of carbohydrates, such as D-glucose, D-galactose, maltose,
cellobiose, lactose, D-glucose 6-phasphate, D-mannose,
2-deoxy-D-glucole, 2-deoxy-D-galactose, D-fucase, D-glucurnic acid,
and D-xylose.
Also the red sea-weed Iridophrycus flaccidum produces easily
extractable hexose oxidases, which oxidise several different mono-
and disaccharides (Bean and Hassid, (1956), J. Biol. Chem, 218, p.
425; Rand et al. (1972, J. of Food Science 37, p. 698-710).
The broad substrate spectrum of hexose oxidase is advantageous in
the connection with tooth bleaching as the total amount of usable
substrate (i. e. carbohydrate) present in the mouth is
significantly greater than for related enzymes having more specific
catalytic properties.
Another relevant group of tooth bleaching enzymes is xylitol
oxidases (see e.g. JP 80892242) which oxidises xylitol, D-sorbitol,
D-galactitol, D-mannitol and D-arabinitol in the presence of
oxygen. A xylitol oxidase can be obtained from strains of
Streptomyces sp. (e.g. Streptomyces IKD472, FERM P-14339) having a
pH optimum at 7.5, is stable at pH 5.5 to 10.5 and at temperatures
up to 65.degree. C.; properties very well suited for oral care
compositions and products. Further, the substrate xylitol is not
cariogenic (i.e. is not degraded in the mouth to compounds
responsible for dental holes).
It can be advantageous to use enzyme(s) which can act on substrates
which are not cariogenic (i.e. substrates which are not or is not
immediately degraded into cariogenic substrates such as sucrose,
glucose, fructose, maltose etc.).
Examples of such a substrate include amino acids, alcohol, sugar
alcohol, such as xylitol, sorbitol etc.
Consequently, in a preferred embodiment the oral care composition
comprises one or more of the above enzymes and a substrate which is
not cariogenic.
It is also advantageous to use enzymes being substantially active
at pHs prevailing in the mouth, i.e. between pH 5.0 to 9.0,
preferably between pH 6.0 to 8.5, especially between pH 6.4 to
7.5.
The term "substantially active" enzyme means in this context that
the enzyme(s) has(have) an relative activity (pH-optimum defines
100% at the same conditions) higher than 30%, better 50%, even
better more than 70%, such as 80%, and in the best case up to about
100% of the activity at the pH optimum.
In the case of using laccase and a peroxidase the bleaching effect
can be obtained by direct oxidation of the teeth stains or via a
mediator.
Oxidases generate peroxide (H.sub.2 O.sub.2) in situ (i.e. in the
oral cavity).
When using laccase and/or peroxidase, or an oxidase and a
peroxidase no peroxide need to be present, while low concentrations
of peroxide need to be added in the case of a peroxidase alone.
However, it is also contemplated according to the invention to add
low concentrations of peroxide when using laccase or oxidase.
It is to be understood that an advantage of the invention is that
peroxide will not at any time be present in the oral cavity in
concentrations which can inflict damage.
Compositions and products of the invention can be used safely
without any major precautions by both the private consumer and
professionals (i.e. dentists or the like) due to the low
concentration or absence of hydrogen peroxide.
In a preferred embodiment the oral composition or oral care product
of the invention comprises a redox mediator (sometimes called an
enhancing agent or accelerator) which is an agent capable of
enhancing the activity of oxidoreductases contemplated according to
the invention.
The mediator may be any known mediator. Examples of such mediators
are the following compounds:
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS);
6-hydroxy-2-naphtoic acid; 7-methoxy-2-naphtol;
7-amino-2-naphthalene sulfonic acid; 5-amino-2-naphthalene sulfonic
acid; 1,5-diaminonaphthalene; 7-hydroxy-1,2-naphthimidazole;
10-methylphenothiazine; 10-phenothiazine-propionic acid (PPT);
N-hydroxysuccinimide-10-phenothiazine-propionate; benzidine;
3,3'-dimethylbenzidine; 3,3'-dimethoxybenzidine;
3,3',5,5'-tetramethylbenzidine; 4'-hydroxy-4-biphenylcarboxylic
acid; 4-amino-4'-methoxystilbene;
4,4'-diaminostilbene-2,2'-disulfonic acid;
4,4'-diaminodiphenylamine; 2,7-diaminofluorene;
4,4'-dihydroxy-biphenylene; triphenylamine;
10-ethyl-4-phenothiazinecarboxylic acid; 10-ethylphenothiazine;
10-propylphenothiazine; 10-isopropylphenothiazine;
methyl-10-phenothiazinepropionate; 10-phenylphenothiazine;
10-allylphenothiazine; 10-phenoxazinepropionic acid (POP);
10-(3-(4-methyl-1-piperazinyl)propyl)phenothiazine;
10-(2-pyrrolidinoethyl)phenothiazine; 10-methylphenoxazine;
iminostilbene;
2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonicacid;
N-benzylidene-4-biphenylamine; 5-amino-2-naphthalenesulfonic acid;
7-methoxy-2-naphtol; 4,4'-dihydroxybenzophenone;
N-(4-(dimethylamino)benzylidene)-p-anisidine;
3-methyl-2-benzothiazolinone(4-(dimethylamino)benzylidene)hydrazone;
2-acethyl-10-methylphenothiazine;10-(2-hydroxyethyl)phenothiazine;10-(2-hy
droxyethyl)phenoxazine;10-(3-hydroxypropyl)phenothiazine;
4,4'-dimethoxy-N-methyl-diphenylamine; vanillin azine.
The amount of oxidoreductase(s) needed in an oral composition of
the invention to obtain tooth bleaching depends on the particular
compound employed, but ranges generally from 0.0001% to 20%,
preferably from about 0.001% to about 10%, and most preferably from
about 0.01% to about 5% by weight of the final composition.
In the preparation of an oral composition, the oxidoreductase(s)
may for safety reasons be added as an essentially purified enzyme
preparation. However, less purified oxidoreductases preparations
can be used seen from a technical point of view.
The oral composition may be incorporated in products used for vital
and/or non-vital tooth bleaching techniques. Examples of products
suitable for vital tooth bleaching, having the main purpose of
bleaching teeth, include products usually used by trained
professionals in-office tooth bleaching and/or in the so-called
"dental prescribed, home-applied"-products.
Especially contemplated according to the invention are
"over-the-counter"-products, which include tooth bleaching kits and
conventional oral care products.
Conventional oral care products are products, such as toothpastes,
gels, mouth washes or denture cleaning agents usually primarily
target dental caries, plaque and/or tartar. However, oral care
products also targeting tooth stains (often called tooth whiteners)
have been available on the consumer market for some years. However,
these products use another bleaching/whitening principle.
The above mentioned types of products are described further in the
above section "Background of the Invention".
The oral composition or oral care product of the invention may
comprise at least one other enzyme activity, which includes the
activity of a protease, and/or mutanase and/or dextranase and/or
lipase and/or amylase and/or anti-microbial polypeptides or
enzymes.
In a preferred embodiment of the invention the oral composition or
oral care product comprise an oxidoreductase and a dextranase
and/or a mutanase.
An oral composition of the invention may advantageously be used for
in conventional oral care products having any suitable physical
form (i.e. powder, paste, gel, liquid, ointment, tablet etc.).
In an embodiments the oral composition or oral care products are
one-compartment products in air-free packages containing an
oxidoreductase enzyme or enzyme system, especially a laccase, a
redox mediator agent (e.g. ABTS or PPT) and further ingredients
normally used in such oral compositions or oral care products.
In another embodiment the oral care compositions or oral care
products are one-compartment products comprising an oxidase and the
corresponding substrate with a limited amount of water or a
none-aqueous dentifrice.
Even though the presence of a redox mediator is advantageous, as it
improves the action of the tooth bleaching, it is not
compulsory.
Also contemplated according to the invention are two-compartment
oral compositions and oral care products, where the oxidoreductase
(e.g. laccase) and the redox mediator are mixed immediately before
introduction into the oral cavity.
An "oral care product" of the invention is defined as a product
which can be used for maintaining and/or improving oral hygiene in
the mouth of humans and animals, and/or preventing or treating
dental diseases.
Examples of such oral care products include toothpaste, dental
cream, gel or tooth powder, odontic, mouth washes, denture cleaning
agents, pre- or post brushing rinse formulations, chewing gum,
lozenges, and candy.
Toothpastes and tooth gels typically include abrasive polishing
materials, foaming agents, flavouring agents, humectants, binders,
thickeners, sweetening agents, and water.
Mouth washes, including plaque removing liquids, typically comprise
a water/alcohol solution, flavour, humectant, sweetener, foaming
agent, and colorant.
According to the invention said abrasive polishing material
includes alumina and hydrates thereof, such as alpha alumina
trihydrate, magnesium trisilicate, magnesium carbonate, sodium
bicarbonate ("Baking soda"), kaolin, aluminosilicates, such as
calcined aluminum silicate and aluminum silicate, calcium
carbonate, zirconium silicate, and also powdered plastics, such as
polyvinyl chloride, polyamides, polymethyl methacrylate,
polystyrene, phenol-formaldehyde resins, melamine-formaldehyde
resins, urea-formaldehyde resins, epoxy resins, powdered
polyethylene, silica xerogels, hydrogels and aerogels and the like.
Also suitable as abrasive agents are calcium pyrophosphate,
water-insoluble alkali metaphosphates, dicalcium phosphate and/or
its dihydrate, dicalcium orthophosphate, tricalcium phosphate,
particulate hydroxyapatite and the like. It is also possible to
employ mixtures of these substances.
Dependent on the oral care product the abrasive product may be
present in from 0 to 70% by weight, preferably from 1% to 70%. For
toothpastes the abrasive material content typically lies in the
range from 10% to 70% by weight of the final toothpaste
product.
Humectants are employed to prevent loss of water from e.g.
toothpastes. Suitable humectants for use in oral care products
according to the invention include the following compounds and
mixtures thereof: glycerol, polyol, sorbitol, polyethylene glycols
(PEG), propylene glycol, 1,3-propanediol, 1,4-butanediol,
hydrogenated partially hydrolysed polysaccharides and the like.
Humectants are in general present from 0% to 80%, preferably 5 to
70% by weight in toothpaste.
Silica, starch, tragacanth gum, xanthan gum, extracts of Irish
moss, alginates, pectin, cellulose derivatives, such as
hydroxyethyl cellulose, sodium carboxymethyl cellulose and
hydroxypropyl cellulose, polyacrylic acid and its salts,
polyvinylpyrrolidone, can be mentioned as examples of suitable
thickeners and binders, which help stabilizing the dentifrice
product. Thickeners may be present in toothpaste creams and gels in
an amount of from 0.1 to 20% by weight, and binders to the extent
from 0.01 to 10% by weight of the final product.
As foaming agent soap, anionic, cationic, non-ionic, amphoteric
and/or zwitterionic surfactants can be used. These may be present
at levels from 0% to 15%, preferably from 0.1 to 13%, more
preferably from 0.25 to 10% by weight of the final product.
Surfactants are only suitable to the extent that they do not exert
an inactivation effect on the present enzymes. Surfactants include
fatty alcohol sulphates, salts of sulphonated mono-glycerides or
fatty acids having 10 to 20 carbon atoms, fatty acid-albumen
condensation products, salts of fatty acids amides and taurines
and/or salts of fatty acid esters of isethionic acid.
Suitable sweeteners include saccharin and/or other appropriate
sweeteners.
Flavours, such as spearmint and peppermint, are usually present in
low amounts, such as from 0.01% to about 5% by weight, especially
from 0.1% to 5%.
Water is usually added in an amount giving e.g. toothpaste a
flowable form, i.e. between 40% to 70% by weight of the final
product.
Further water-soluble anti-bacterial agents, such as chlorhexidine
digluconate, hexetidine, alexidine, quaternary ammonium
anti-bacterial compounds and water-soluble sources of certain metal
ions such as zinc, copper, silver and stannous (e.g. zinc, copper
and stannous chloride, and silver nitrate) may also be
included.
Also contemplated according to the invention is the addition of
anti-calculus agents, anti-plaque agents, compounds which can be
used as fluoride source, dyes/colorants, preservatives, vitamins,
pH-adjusting agents, anti-caries agents, desensitizing agents
etc.
A toothpaste produced from an oral composition of the invention (in
weight % of the final toothpaste composition) may e.g. comprise the
following ingredients:
______________________________________ Abrasive material 10 to 70%
Humectant 0 to 80% Thickener 0.1 to 20% Binder 0.01 to 10%
Sweetener 0.1% to 5% Foaming agent 0 to 15% Bleaching enzyme
0.0001% to 20% Other enzymes 0 to 20% Peroxide 0 to 1%
______________________________________
Mouth washes
A mouth wash produced from an oral care composition of the
invention (in weight % of the final mouth wash composition) may
typically comprise the following ingredients:
______________________________________ 0-20% Humectant 0-2%
Surfactant 0-5% Enzymes 0-20% Ethanol 0-2% Other ingredients (e.g.
flavour, sweetener active ingredients such as florides). 0-70%
Water ______________________________________
The mouth wash composition may be buffered with an appropriate
buffer e.g. sodium citrate or phosphate in the pH-range 6-7.5.
The mouth wash may be in none-diluted form (i.e. must be diluted
before use).
Method of Manufacture
The oral care composition and products of the present invention can
be made using methods which are common in the oral product
area.
Finally the invention relates to a method for using an oral care
product of the invention, wherein
a) the oral care product is introduced into the mouth,
b) contacted with the teeth and/or gums for a period of time,
c) removed from the mouth, and
d) optionally rinsed with a liquid.
If the oral care product to be used is in solid to flowable form a
tooth brush or the like may advantageously be used for contacting
the oral care product with the teeth and/or gums. In the case of a
liquid oral care product the contact may take place by rinsing the
mouth.
The time period of contact in step b) is optional. However,
contacting the oral care product with the teeth and/or gums for
between about 30 seconds to 15 minutes will normally be sufficient
for obtaining the desired result.
After use, the oral care product may be removed from the mouth in
any suitable way, e.g. by spitting it out. Optionally the mouth may
be rinsed with a liquid, such as tap water.
METHODS AND MATERIALS
Materials:
Enzyme:
Laccase from Myceliopthora thermophila, (available from Novo
Nordisk A/S).
Glucose oxidase from Aspergillus niger (available from Novo Nordisk
A/S).
L-amino acid oxidase from Trichoderma harzianum (available from
Novo Nordisk A/S).
Teeth:
Alike coloured teeth for the bleaching tests are selected by colour
determination on the Minolta CR-221 Chroma Meter. The teeth are
stored in water under refrigeration until use.
Solutions:
______________________________________ Staining broth A: (extrinsic
stains) Clarified saliva* 10 ml Tea** 10 ml Coffee*** 10 ml Milk 10
ml ______________________________________ *Clarified salvia is
prepared by the following steps: chewing parafilm and collecting
saliva, centrifuging it and collecting the supernatant **Pour 50 ml
of boiled water on to one Lipton .TM. Yellow label tea bag and
leave it for 10 minutes. ***Regular coffee
Staining broth B: (extrinsic stains) 100 ml autoclaved TSB (Tryptic
Soy broth) 0.35 g instant coffee powder (Nescafe .TM., Classic)
0.25 g gastric mucin Tea extract* 3.25 ml TSB broth with 24 hours
aerobicly grown Micrococcus luteus DSM 20030T culture (from
Nutrient agar plates grown for 2 days at 37.degree. C.). *40 ml
boiling water is poured to tea from one bag of Lipton .TM. Yellow
lable tea bag and are allowed to stand for 2 minutes. 15 ml of the
supernatant is filtrated with a sterilized membrane, 0.45 .mu..
______________________________________ Straining broth C:
(intrinsic stains) 2.5% BSA in 0.1M phosphate buffer, pH 7. 10 ml
0.1M Phosphate buffer, pH 7 2.5 ml 0.2% chlorhexidine digluconate
25 ml 30% ribose 12.5 ml Incubate at 37.degree. C.
______________________________________
Equipment:
Chroma Meter 210 (Minolta)
Methods:
Preparation of hydroxyapatite tablets
Hydroxyapatite tablets are prepared by compressing 250 mg of
hydroxyapatite in a tablet die at about 5,900 kg (13,000 lbs) of
pressure for 5 minutes. The tablets are then sintered at
600.degree. C. for 4 hours and finally hydrated with sterile
de-ionised water.
Sterilization of hydroxyapatite tablets
HA tablets are sterilised at 180.degree. C. for two hours, hydrated
with the sterilised de-ionised water and placed in a lid of Nunc
tube (10 ml volume).
Staining of bovine teeth:
Bovine teeth are stained by immersing in staining broth A at
37.degree. C until teeth surface become brown.
HA tablets stained with pellicle
HA tablets are immersed in 2 ml of the staining broth B placed in a
Nunc tube, rotated with approximately 30.degree. angle for 5
seconds with an interval of 10 seconds at 37.degree. C. The
staining broth is replaced every 24 hours. The HA tablets are
incubated for 3 days.
Assessment of the teeth colour
Quantitative teeth color assessment.
The color of teeth and hydroxyapatite tablets are measured before
and after the enzyme treatment on a Chroma Meter 210. The Chroma
Meter has a 3 mm diameter circular aperture for measuring optical
properties of small areas. The meter provides values of the optical
parameters L*, a* and b* in the CIE system of colour measurement.
.DELTA.L* is the difference of L* before and after a treatment
which relates to the overall lightness or darkness change. Total
colour difference is given by .DELTA.E* calculated with an equation
below. A high positive value of .DELTA.L* , indicating lightness.
##EQU1##
The color of teeth should be measured after significant incubation
of teeth in de-ionised water (at least 15 minutes at room
temperature). The surface of teeth are wiped lightly with paper
towel and the colour is measured.
L*: "0"=black and "100"=white
Determination of Laccase Activity (LACU)
Laccase activity is determined from the oxidation of syringaldazin
under aerobic conditions. The violet colour produced is
photometered at 530 nm. The analytical conditions are 19 .mu.M
syringaldazin, 23.2 mM acetate buffer, pH 5.5, 30.degree. C., 1
minute reaction time.
1 laccase unit (LACU) is the amount of enzyme that catalyses the
conversion of 1.0 .mu.mole syringaldazin per minute under these
conditions.
Determination of peroxidase activity units (POXU)
Peroxidase activity is measured in POXU/ml. (1 POXU (peroxidase
unit) is defined as the amount of enzyme that catalyses the
conversion of 1 .mu.mole H.sub.2 O.sub.2 per minute in a system
where 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonate] is oxidised
in the presence of 1 mM H.sub.2 O.sub.2, pH 7.0, at a temperature
of 40.degree. C.)
Determination of glucose oxidase activity (GODU)
1 GODU is defined as the amount of enzyme which, under standard
conditions, catalyses the formation of 1 micromole of H.sub.2
O.sub.2 per minute. The analytic method AF266 is available upon
request from Novo Nordisk A/S).
EXAMPLES
Example 1
Bleaching of Extrinsic Teeth Stains with Glucose Oxidase (GOX)
Bovine teeth were immersed in staining broth A comprising tea,
coffee, milk, and saliva at 37.degree. C. until the teeth surface
became brown as described in the Materials and Methods. section and
treated with solution A and B, and Buffer as described below.
______________________________________ Solution A Solution B Enzyme
Glucose Buffer ______________________________________ 1 none none
(Buffer) 50 mM Britton- Robinson pH 6.5 2 Glucose oxidase 500 mM
glucose 50 mM Britton- 50 GODU/ml Robinson pH 6.0
______________________________________ Both samples were prepared
with BrittonRobinson buffer (pH 6.5) Glucose solution was prepared
one day before and kept at 4.degree. C. The glucose oxidase was
derived from Aspergillus niger
Each solution was pre-incubated at 37.degree. C. for 15 minutes
prior to experiments
Two teeth were placed in a tube with 5.0 ml of solution A and 5.0
ml of solution B, respectively and incubation for 60 minutes at
37.degree. C. A small magnetic stirrer was used for stirring the
solutions. Both samples with buffer and oxidase+glucose,
respectively, were treated this way. The treatment was repeated 3
times.
After incubation, the teeth were gently rinsed and the colour was
measured as described in the Materials and Method section.
The result of the test is displayed in Table 1
TABLE 1 ______________________________________ .DELTA.L* .DELTA.E*
______________________________________ Buffer -0.15 3.13 25 GODU/ml
glucose oxidase + 0.74 1.80 250 mM glucose
______________________________________
As can be seen from the Table 1 glucose oxidase is capable of
bleaching teeth with extrinsic stains.
Example 2
Bleaching of Extrinsic Stained Hydroxy Apatite Tablets with L-amino
Acid Oxidase
Hydroxyapatite (HA) tablets were extrinsicly stained according to
Stookey's method (Stookey G. K. et al. "In vitro Removal of Stains
with Dentifrices", J. Dent. Res. 61, p. 1236 (1982)).
HA tablets (mimicking the properties of human enamel) sterilized as
described in the Material and Methods section were immersed in
staining broth B consisting of tea, coffee, mucin and Micrococcus
luteus for 5 seconds with 10 seconds intervals at 37.degree. C. as
described above in the Materials and Method section. The HA tablets
were incubated for 3 days. After staining the HA tablets were air
dried and washed with de-ionised water
Chromatographically pure L-amino acid oxidase from Trichoderma
harzianum free from catalase activity was incubated with a
L-Arginine solution prepared in 0.1 M sodium phosphate buffer pH
8.5.
Two stained HA tablets were treated with said L-amino acid oxidase
solution by incubating in a total volume of 0.5 ml at 37.degree. C.
for 60 minutes with 90 reciprocal shaking/minutes.
The optical parameters L*, a* and b* were measured on a Chroma
Meter as described in the Materials and Methods section.
The result of the test in displayed in Table 2
TABLE 2 ______________________________________ .DELTA.L* .DELTA.E*
______________________________________ Buffer -0.17 0.30 6 Units/ml
L-amino acid oxidase + 100 1.04 1.07 mM L-Arginine
______________________________________
As can be seen from the Table 2 L-amino acid oxidase is capable of
bleaching teeth with extrinsic stains.
Example 3
Bleaching of Extrinsic Stained Hydroxy Apatite Tablets with Glucose
Oxidase
Hydroxyapatite (HA) tablets were stained according to Stookey's
method (Stookey G. K. et al. "In vitro Removal of Stains with
Dentifrices ", J. Dent. Res. 61, p. 1236 (1982)) and treated with
chromatographically pure glucose oxidase from Aspergillus niger,
free from catalase activity, incubated with a glucose solution
prepared in 20 mM Britton-Robinson buffer, pH 6.0.
One stained HA tablet was treated with said glucose oxidase
solution by incubating in a total volume of 10 ml at 39.degree. C.
for 60 minutes with 90 reciprocal shaking/minutes.
The optical parameters L*, a* and b* were measured on a Chroma
Meter as described in the Materials and Methods section.
The result of the test in displayed in Table 3
TABLE 3 ______________________________________ .DELTA.L* .DELTA.E*
______________________________________ Buffer 2.18 2.97 500 mM
glucose 1.80 1.84 10 GODU/ml glucose oxidase + 3.50 3.73 100 mM
glucose 10 GODU/ml glucose oxidase + 3.62 3.62 500 mM glucose 50
GODU/ml glucose oxidase + 3.90 4.83 500 mM glucose 100 GODU/ml
glucose oxidase + 5.05 5.63 500 mM glucose
______________________________________
As can be seen from the Table 3 glucose oxidase is capable of
bleaching teeth with extrinsic stains.
Example 4
Bleaching of Extrinsic and Intrinsic Teeth Stains with Laccase
Bovine teeth were immersed in the staining broth (extrinsic stains)
overnight at 37.degree. C., transferred to staining broth c
(intrinsic stains) and incubated at 37.degree. C. until the tooth
surface became brown.
______________________________________ Solution A Solution B Enzyme
mediator ______________________________________ 1 none (Buffer)
none (Buffer) 2 0.6 LACU/ml Laccase PTT 1000 .mu.M
______________________________________ Samples were all prepared
with 50 mM BrittonRobinson buffer pH 6.5 The laccase was derived
from Myceliophthora thermophila
Each of solutions A and B was pre-incubated at 37.degree. C. for 15
minutes prior to experiments
Two teeth were placed in a tube with 5.0 ml of solution A and 5.0
ml of solution B, respectively, and incubation for 60 minutes at
37.degree. C. A small magnetic stirrer was used for stirring the
samples. Both samples with buffer and Laccase+PPT, respectively,
were treated this way. The treatment was repeated 3 times for each
sample.
After incubation, the teeth were gently rinsed and the colour was
measured as described in the Materials and Method section.
The result of the test is displayed in Table 4
TABLE 4 ______________________________________ .DELTA.L* .DELTA.E*
______________________________________ Buffer 0.01 1.53 0.3 LACU/ml
laccase + 500 .mu.M PPT 2.67 2.78
______________________________________
As can be seen from the Table 4 laccase is capable of bleaching
teeth with intrinsic stains. As will be apparent to those skilled
in the art in the light of the foregoing disclosure, many
alterations and modifications are possible in the practice of this
invention without departing from the spirit or scope thereof.
Accordingly, the scope of the invention is to be construed in
accordance with the substance defined by the following claims.
* * * * *